One-step preparation of cyclen-containing hydrophilic polymeric monolithic materials via epoxy-amine ring-opening reaction and their application in enrichment of N-glycopeptides.

Considering the special structure of 1,4,7,10-tetraazacyclododecane (cyclen) which is easy to form complexes with ions, it is beneficial to achieve particular selectivity. Cyclen was selected as a precursor to react with triglycidyl isocyanurate (TGIC), and a novel kind of hydrophilic polymeric monolithic material was facilely prepared via epoxy-amine ring-opening reaction in the presence of a binary porogenic system of acetonitrile (ACN) and polyethylene glycol. The resulting poly (TGIC-co-cyclen) monolithic column was used to separate both nonpolar alkylbenzenes using mobile phase of ACN/H2O (35/65, v/v) and polar phenolic compounds and anilines under the mobile phase of ACN/H2O (60/40, v/v) in reversed-phase capillary liquid chromatography (cLC). It should be pointed that the monolith was further used for separation of a mixture of toluene, DMF, acrylamide and thiourea under the mobile phase of ACN/H2O (95/5, v/v) by hydrophilic interaction chromatography (HILIC). These results indicated that the poly (TGIC-co-cyclen) column exhibited mixed-mode retention mechanism. As a result, the prepared monolithic material was employed for enrichment of glycosylated peptides from the tryptic digest of human immunoglobulin G (IgG) and serum protein tryptic digests. A total of 531 N-glycopeptides and 329 N-glycosylation sites, mapped to 166 glycoproteins, were identified from 2 µL human serum digest. The results indicated the prepared monolith had ability for enriching N-glycopeptides from complex biological samples.

Enantiomer separation on novel cellulose derivatives bearing regioselective phenylcarbamate groups.

Four novel regioselectively carbamoylated cellulose derivatives with two types of substituents, one being a 3-chloro-4-methylphenylcarbamate group and the other being a phenylcarbamate, a 3,5-dichlorophenylcarbamate or a 4-methylphenylcarbamate group, at the 2-, 3-positions and 6-position were synthesized and utilized as chiral stationary phases in high-performance liquid chromatography. The obtained derivatives exhibited characteristic chiral recognition abilities which were significantly affected by the nature and arrangement of the groups at the 2-, 3-positions and 6-position. Compared with cellulose tris(3-chloro-4-methylphenylcarbamate) and Chiralcel OD columns, several racemates were better resolved on these new phases. With all six racemates resolved on cellulose 2,3-bis(4-methylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate), it appears to be a potential useful chiral stationary phase in the future. Also, the influence of mobile phase on chiral recognition was investigated. Better resolutions were generally obtained with decreased amount of 2-propanol with a mixture of hexane/2-propanol as the mobile phase. Moreover, when ethanol was used instead of 2-propanol as the mobile phase modifier, poorer enantioseparations were often achieved.

[Preparation and chiral recognition of heterosubstituted amylose derivatives-based chiral stationary phases].

Two novel amylose derivatives, namely amylose 2-benzoate-3-(4-methylphenylcarbamate)-6-(3,5-dichlorophenylcarbamate) and amylose 2-benzoate-3-(3,5-dichlorophenylcarbamate)-6-(4-methylphenylcarbamate), were prepared utilizing a serial regioselective process. After coated onto aminopropyl silica gel, they were utilized as chiral stationary phases (CSPs) for high performance liquid chromatography. Investigations indicated that the CSPs exhibited characteristic chiral recognition and their chiral recognition abilities were much higher than those of amylose tris(3,5-dichlorophenylcarbamate) (ADCPC), a homosubstituted derivative. The nature and position of the substituents at 3-, and 6-positions of a glucose unit had great influence on the chiral resolution abilities of the amylose derivatives. Some chiral compounds which were not effectively resolved on the commercial column Chiralpak AD were effectively separated on the new CSPs. Moreover, with all the eight tested racemates resolved, amylose 2-benzoate-3-(4-methylphenylcarbamate)-6-(3,5-dichlorophenylcarbamate) exhibited relatively high chiral recognition and might be a potential useful CSP.

Preparation and evaluation of regioselectively substituted amylose derivatives for chiral separations.

Six novel regioselectively substituted amylose derivatives with a benzoate at 2-position and two different phenylcarbamates at 3- and 6-positions were synthesized and their structures were characterized by 1 H nuclear magnetic resonance (NMR) spectroscopy. Their enantioseparation abilities were then examined as chiral stationary phases (CSPs) for high-performance liquid chromatography (HPLC) after they were coated on 3-aminopropyl silica gels. Investigations indicated that the substituents at the 3- and 6-positions played an important role in chiral recognition of these amylose 2-benzoate serial derivatives. The derivatives demonstrated characteristic enantioseparation and some racemates were better resolved on these derivatives than on Chiralpak AD, which is one of the most efficient CSPs, utilizing coated amylose tris(3,5-dimethylphenylcarbamate) as the chiral selector. Among the derivatives prepared, amylose 2-benzoate-3-(phenylcarbamate/4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) exhibited chiral recognition abilities comparable to that of Chiralpak AD and may be useful CSPs in the future. The effect of mobile phase on chiral recognition was also studied. In general, with the decreased concentration of 2-propanol, better resolutions were obtained with longer retention times. Moreover, when ethanol was used instead of 2-propanol, poorer resolutions were often achieved. However, in some cases, improved enantioselectivity was achieved with ethanol rather than 2-propanol as the mobile phase modifier.

Facile preparation of a stable and functionalizable hybrid monolith via ring-opening polymerization for capillary liquid chromatography.

An organic-inorganic hybrid monolith was prepared by a single-step ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS) with poly(ethylenimine) (PEI). The obtained hybrid monoliths possessed high ordered 3D skeletal microstructure with dual retention mechanism that exhibits reversed-phase (RP) mechanism under polar mobile phase and hydrophilic-interaction liquid chromatography (HILIC) retention mechanism under less polar mobile phase. The high column efficiencies of 110,000N/m can be achieved for separation of alkylbenzenes in capillary reversed-phase liquid chromatography (cLC). Due to the robust property of hybrid monolith and the rich primary and secondary amino groups on its surface, the resulting hybrid monolith was easily modified with γ-gluconolactone and physically coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), respectively. The former was successfully applied for HILIC separation of neutral, basic and acidic polar compounds as well as small peptides, and the latter for enantioseparation of racemates in cLC. The high column efficiencies were achieved in all of those separations. These results demonstrated that the hybrid monolith (POSS-PEI) possessed high stability and good surface tailorbility, potentially being applied for other research fields.

Hybrid monolithic columns coated with cellulose tris(3,5-dimethylphenyl-carbamate) for enantioseparations in capillary electrochromatography and capillary liquid chromatography.

A hybrid monolithic capillary column synthesized with (3-chloropropyl)-trimethoxysilane (CPTMS) and tetramethoxysilane (TMOS) via sol-gel chemistry was in situ coated with cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) for enantioseparations in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC). Prior to coating, the prepared CP-silica hybrid monolith was straightforwardly modified with diethylenetriamine (DETA) to introduce NH(2) functionalities via the nucleophilic substitution reaction, which generate the stronger EOF for CEC. The coating condition was optimized to obtain a stable and reproducible chiral stationary phase for enantioseparation. The results indicated that racemic benzoin was baseline separated on the resulting hybrid monolith coated with 30 mg/mL CDMPC in CEC, while several racemates were successfully enantioseparated on the resulting CP-silica hybrid monolithic column coated with 60 mg/mL CDMPC in CLC with RP and NP modes.

Synthesis and HPLC chiral recognition of regioselectively carbamoylated cellulose derivatives.

Four regioselective-carbamoylated cellulose derivatives having two different substituents at 2-, 3-, and 6-position were prepared and evaluated as chiral stationary phases (CSPs) for high-performance liquid chromatography. Investigations showed that the nature and arrangement of the substituents significantly influenced the chiral recognition abilities of the heterosubstituted cellulose derivatives and each derivative exhibited characteristic enantioseparation. Some racemates were better resolved on these derivatives than the corresponding homogeneously substituted cellulose derivatives including a commercial CSP, Chiralcel OD. Racemic compounds shown in this study were most effectively discriminated on cellulose 2,3-(3-chloro-4-methylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate) and 2,3-(3,5-dimethylphenylcarbamate)-6-(3-chloro-4-methylphenylcarbamate).

Improved preparation of chiral stationary phases via immobilization of polysaccharide derivative-based selectors using diisocyanates.

The classical method for the preparation of immobilized polysaccharide-based chiral stationary phases (CSPs) with a diisocyanate was improved. Cellulose or amylose was directly coated onto 3-aminopropyl silica gel after it was dissolved in a mixture of N,N-dimethylacetamide, LiCl, and pyridine, then immobilized onto silica gel with a diisocyanate, and finally allowed to react with an excess of corresponding isocyanate. Four polysaccharide derivatives, 3,5-dimethylphenylcarbamate and 3,5-dichlorophenylcarbamate of cellulose, and 3,5-dimethylphenylcarbamate and 5-chloro-2-methylphenylcarbamate of amylose, were immobilized onto silica gel utilizing this method. Compared with the classical diisocyanate method, the improved procedure avoided the derivatization and regeneration of 6-hydroxyl groups of cellulose and amylose, and thus showed an advantage for simple and economical preparation. The relationships among the amount of diisocyanate used, immobilization efficiency, and enantioseparation on the cellulose-based CSPs were investigated. Also, the solvent durability of the obtained CSPs was examined with eluents containing chloroform or THF. By utilizing these eluents, the chiral recognition abilities of the obtained CSPs for some of the tested racemates were improved.

[Preparation and evaluation of amylose and cellulose tris (3-trifluoromethylphenylcarbamates)-based chiral stationary phases].

To broaden the category of polysaccharide-based chiral stationary phases (CSPs), coated CSPs based on tris (3-trifluoromethylphenylcarbamates) of amylose and cellulose were prepared for enantioseparation in high performance liquid chromatography. Their performances were evaluated by eight racemates using hexane-isopropanol as mobile phase. Compared with the most widely used, commercially available polysaccharide-based CSPs, Chiralpak AD and Chiralcel OD, utilizing tris (3,5-dimethylphenylcarbamates) of amylose and cellulose as the chiral selector, respectively, the obtained CSPs exhibited lower enantioseparation abilities. However, cellulose tris(3-trifluoromethylphenylcarbamate)-based CSP exhibited characteristic enantioseparation and some chiral compounds were better resolved on this CSP than on Chiral-cel OD. The chiral recognition abilities of the obtained CSPs were increased with the decrease of the portion of isopropanol in the mobile phase and relatively high enantioseparation was obtained with the mobile phase of hexane-isopropanol (95: 5, v/v). The obtained amylosic phase demonstrated slightly higher chiral resolving ability than the cellulosic one for the test racemates. In addition, it was revealed that the enantioseparations of the cellulosic and amylosic CSPs were complementary.

Immobilization of 3,5-dimethylphenylcarbamates of cellulose and amylose onto silica gel using (3-glycidoxypropyl)triethoxysilane as linker.

The 3,5-dimethylphenylcarbamates of cellulose and amylose were effectively immobilized onto plain silica gels as chiral packing materials (CPMs) for HPLC by means of intermolecular polycondensation of triethoxysilyl groups introduced with (3-glycidoxypropyl)triethoxysilane. The immobilization and chiral recognition abilities of the obtained CPMs prepared with different amounts of (3-glycidoxypropyl)triethoxysilane were investigated. In addition, the solvent compatibilities of the immobilized-type CPMs were examined with eluents containing chloroform and THF. When these eluents were used, for most of the tested racemates, the chiral resolving abilities of the obtained CPMs were improved.

Immobilization and chiral recognition of 3,5-dimethylphenylcarbamates of cellulose and amylose bearing 4-(trimethoxysilyl)phenylcarbamate groups.

A small amount of 4-(trimethoxysilyl)phenyl groups was randomly introduced onto the 3,5-dimethylphenylcarbamates of cellulose and amylose by a one-pot method. The obtained derivatives were then effectively immobilized onto silica gel as chiral packing materials (CPMs) for high-performance liquid chromatography through intermolecular polycondensation of the trimethoxysilyl groups. The effects of the amount of 4-(trimethoxysilyl)phenyl groups on immobilization and enantioseparation were investigated. Also, the solvent durability of the immobilized-type CPMs was examined with the eluents containing chloroform and tetrahydrofuran. When these eluents were used, the chiral recognition abilities of the CPMs for most of the tested racemates were improved to some extent depending on the compounds.

Immobilization of cellulose phenylcarbamate onto silica gel via in termolecular polycondensation of triethoxysilyl groups introduced with (3-glycidoxypropyl)triethoxysilane.

Cellulose 3,5-dimethylphenylcarbamate was successfully immobilized onto bare silica gel for HPLC through the intermolecular polycondensation of triethoxysilyl groups, which were introduced onto the cellulose derivative via epoxide ring-opening reaction under acidic conditions. The immobilized-type chiral packing material (CPM) exhibited high chiral recognition ability and could be used with various eluents, which are incompatible with the conventional CPMs prepared by coating the derivative onto silica gel.

Application of cross-linked beta-cyclodextrin polymer for adsorption of aromatic amino acids.

Beta-cyclodextrin (beta-CyD) was cross-linked by hexamethylene diisocyanate and the polymer was investigated for adsorption of aromatic amino acids (AAA) from phosphate buffer. High adsorption rates were observed at the beginning and the adsorption equilibrium was then gradually achieved in about 45 min. The adsorption of AAA decreased with the increase of initial concentration and also temperature. Under the same conditions, the adsorption efficiencies of AAA were in the order of L-tryptophan (L-Trp) > L-phenylalanine (L-Phe) > L-tyrosine (L-Tyr). Much higher adsorption values, up to 52.4 and 43.0 mg/g for L-Trp and L-Phe, respectively, at 50 mmol/L and 3.2 mg/g for L-Tyr at 2 mmol/L, were obtained with the beta-CyD polymer at 37 degrees C. It was shown that the adsorption of AAA on the beta-CyD polymer was consistent with the Freundlich isotherm equation. The adsorption of mixed aromatic amino acids and branched-chain amino acids (BCAA) showed that AAA were preferentially adsorbed with adsorption efficiencies 10-24%, while those of BCAA were lower than 2%. It seems that the structure and hydrophobicity of amino acid molecules are responsible for the difference in adsorption, by influencing the strength of interactions between amino acid molecule and the polymer.

Chiral separation of 1,1'-bi-2-naphthol and its analogue on molecular imprinting monolithic columns by HPLC.

Two molecular imprinting polymer (MIP) monolithic columns with (S)-(-)-1,1'-bi-2-naphthol and (R)-(+)-5,5',6,6',7,7',8,8'-octahydro-1,1'-bi-2-naphthol as the templating molecules, respectively, have been prepared by in situ polymerization using 4-vinylpyridine and ethylene dimethacrylate as functional monomer and cross-linker, respectively. The columns with good flow-through properties were obtained by changing the molar ratio of the functional monomer and the template molecule. The effects of mobile-phase composition on separation of enantiomers were systematically investigated. The results indicate that hydrophobic interaction in aqueous solution and hydrogen-bonding interaction in ACN between the enantiomers and polymers could play important roles in the retention and resolution. The effects of chromatographic conditions, such as flow rate, column temperature, sample loading, on the enantioseparation were also studied. Further, these two MIP columns show a cross-reactivity.